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Summary Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Q o binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies. [ABSTRACT FROM AUTHOR]

Within living cells, mitochondria are considered relevant sources of reactive oxygen species (ROS) and are exposed to reactive nitrogen species (RNS). During the last decade, accumulating evidence suggests that mitochondrial (dys)function, ROS/RNS levels, and aberrations in mitochondrial morphology are interconnected, albeit in a cell- and context-dependent manner. Here it is hypothesized that ROS and RNS are involved in the short-term regulation of mitochondrial morphology and function via non-transcriptional pathways. We review the evidence for such a mechanism and propose that it allows homeostatic control of mitochondrial function and morphology by redox signaling. [ABSTRACT FROM AUTHOR]

Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These “mitochondrial dynamics” are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δ ψ ). The latter is considered a functional readout of mitochondrial “health” and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δ ψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δ ψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondrial NADH:ubiquinone oxidoreductase or complex I (CI) is a frequently affected enzyme in cases of mitochondrial disorders. However, the cytopathological mechanism of the associated pediatric syndromes is poorly understood. Evidence in the literature suggests a connection between mitochondrial metabolism and morphology. Previous quantitative analysis of mitochondrial structure in cultured fibroblasts of 14 patients revealed that mitochondria were fragmented and/or less branched in patients with severe CI deficiency. These patient cells also displayed greatly increased levels of reactive oxygen species (ROS) and marked aberrations in mitochondrial and cellular Ca2+/ATP handling upon hormone stimulation. Here, we discuss the interrelationship between these parameters and demonstrate that the hormone-induced increase in mitochondrial Ca2+ and ATP concentration, as well as the rate of cytosolic Ca2+ removal, are not related to mitochondrial length and/or degree of branching, but decrease as a function of the number of mitochondria per cell. This suggests that the amount of mitochondria, and not their shape, is important for Ca2+-induced stimulation of mitochondrial ATP generation to feed cytosolic ATP-demanding processes. [Copyright &y& Elsevier]

Abstract: Within cells, functional changes in mitochondrial metabolic state are associated with alterations in organelle mobility, shape, and configuration of the mitochondrial matrix. Fluorescence correlation spectroscopy (FCS) is a technique that measures intensity fluctuations caused by single fluorescent molecules moving through a small detection volume. By mathematically correlating these fluctuations, information can be obtained about the concentration and rate of diffusion of the fluorescent molecules. Here we present an FCS‐based approach for determining the mobility of enhanced yellow fluorescent protein (mitoEYFP) in the mitochondrial matrix of primary human skin fibroblasts. This protocol allows simultaneous quantification of intramatrix EYFP concentration and its diffusion constant, as well as the fraction of moving mitochondria and their velocity. [Copyright &y& Elsevier]

Summary: Background & aims: Lipid-induced immune modulation might contribute to the increased infection rate that is observed in patients using parenteral nutrition. We previously showed that emulsions containing medium-chain triglycerides (LCT/MCTs or pure MCTs), but not pure long-chain triglycerides (LCTs), impair neutrophil functions, modulate cell-signaling and induce neutrophil activation in vitro. It has recently been shown that medium-chain fatty acids are ligands for GPR84, a pertussis toxin (PT)-sensitive G-protein-coupled receptor (GPCR). This finding urged us to investigate whether MCT-induced neutrophil activation is mediated by PT-sensitive GPCRs. Methods: Neutrophils isolated from blood of healthy volunteers were pre-incubated with PT (0.5–1μg/mL, 1.5h) and analyzed for the effect of this pre-incubation on LCT/MCT (2.5mmol/L)-dependent modulation of serum-treated zymosan (STZ)-induced intracellular Ca2+ mobilization and on LCT/MCT (5mmol/L)-induced expression of cell surface adhesion (CD11b) and degranulation (CD66b) markers and oxygen radical (ROS) production. Results: PT did not inhibit the effects of LCT/MCT on the STZ-induced increase in cytosolic free Ca2+ concentration. LCT/MCT increased ROS production to 146% of unstimulated cells. However, pre-incubation with PT did not inhibit the LCT/MCT-induced ROS production. Furthermore, the LCT/MCT-induced increase in CD11b and CD66b expression (196% and 235% of unstimulated cells, respectively) was not inhibited by pre-incubation with PT. Conclusion: LCT/MCT-induced neutrophil activation does not involve the action of a PT-sensitive G-protein-coupled receptor. [Copyright &y& Elsevier]

Summary: NADH:ubiquinone oxidoreductase or complex I is a large multisubunit assembly of the mitochondrial inner membrane that channels high-energy electrons from metabolic NADH into the electron transport chain (ETC). Its dysfunction is associated with a range of progressive neurological disorders, often characterized by a very early onset and short devastating course. To better understand the cytopathological mechanisms of these disorders, we use live cell luminometry and imaging microscopy of patient skin fibroblasts with mutations in nuclear-encoded subunits of the complex. Here, we present an overview of our recent work, showing that mitochondrial membrane potential, Ca2+ handling and ATP production are to a variable extent impaired among a large cohort of patient fibroblast lines. From the results obtained, the picture emerges that a reduction in cellular complex I activity leads to a depolarization of the mitochondrial membrane potential, resulting in a decreased supply of mitochondrial ATP to the Ca2+-ATPases of the intracellular stores and thus to a reduced Ca2+ content of these stores. As a consequence, the increase in cytosolic Ca2+ concentration evoked by a Ca2+ mobilizing stimulus is decreased, leading to a reduction in mitochondrial Ca2+ accumulation and ensuing ATP production and thus to a hampered energization of stimulus-induced cytosolic processes. [Copyright &y& Elsevier]

Abstract: Video-rate confocal microscopy of Indo-1-loaded human skeletal myotubes was used to assess the relationship between the changes in sarcoplasmic ([Ca2+]S) and nuclear ([Ca2+]N) Ca2+ concentration during low- and high-frequency electrostimulation. A single stimulus of 10ms duration transiently increased [Ca2+] in both compartments with the same time of onset. Rate and amplitude of the [Ca2+] rise were significantly lower in the nucleus (4.0- and 2.5-fold, respectively). Similarly, [Ca2+]N decayed more slowly than [Ca2+]S (mono-exponential time constants of 6.1 and 2.5s, respectively). After return of [Ca2+] to the prestimulatory level, a train of 10 stimuli was applied at a frequency of 1Hz. The amplitude of the first [Ca2+]S transient was 25% lower than that of the preceding single transient. Thereafter, [Ca2+]S increased stepwise to a maximum that equalled that of the single transient. Similarly, the amplitude of the first [Ca2+]N transient was 20% lower than that of the preceding single transient. In contrast to [Ca2+]S, [Ca2+]N then increased to a maximum that was 2.3-fold higher than that of the single transient and equalled that of [Ca2+]S. In the nucleus, and to a lesser extent in the sarcoplasm, [Ca2+] decreased faster at the end of the stimulus train than after the preceding single stimulus (time constants of 3.3 and 2.1s, respectively). To gain insight into the molecular principles underlying the shaping of the nuclear Ca2+ signal, a 3-D mathematical model was constructed. Intriguingly, quantitative modelling required the inclusion of a satiable nuclear Ca2+ buffer. Alterations in the concentration of this putative buffer had dramatic effects on the kinetics of the nuclear Ca2+ signal. This finding unveils a possible mechanism by which the skeletal muscle can adapt to changes in physiological demand. [Copyright &y& Elsevier]

Determines the structural composition of the functional sodium chloride cotransporter (NCC). Co-immunoprecipitation of NCC subunits; Capability of NCC proteins to form oligometric structures; Expression and function of NCC concatamer in oocytes; Implication on the reabsorption of the filtered load of sodium chloride in the kidney.

Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondria-targeted variants of gallic acid (AntiOxBEN 2) and caffeic acid (AntiOxCIN 4). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 ~23 μM). At a relatively high but non-toxic concentration (12.5 μM), AntiOxBEN 2 and AntiOxCIN 4 increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN 2 and AntiOxCIN 4 did not alter mitochondrial membrane potential (Δψ), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P)H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN 2 and AntiOxCIN 4 both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN 2 (but not AntiOxCIN 4) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN 2 and AntiOxCIN 4 increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN 2 and AntiOxCIN 4 can act as prooxidants thereby activating endogenous ROS-protective pathways. Image 1 • Mitochondriotropic phenolic agents dose-dependently reduced cell viability. • Non-toxic concentrations of AntiOxBEN 2 and AntiOxCIN 4 slightly increased HEt- and CM-H 2 DCF-oxidation but unaltered ΔΨ. • Non-toxic concentrations of AntiOxBEN 2 and AntiOxCIN 4 display prooxidant effects to increase NRF2, SOD2 and GSH levels. • This act as homeostatic circuits to balance ROS detoxification and production, thereby preventing ROS-induced cell death. [ABSTRACT FROM AUTHOR]

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP+) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C 10 -TPP+). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7 -V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety. [Display omitted] • DecylTPP lowers the level of complex I and electron transport chain supercomplexes. • DecylTPP alters mitochondrial membrane potential and size. • DecylTPP reduces mitochondrial oxygen consumption. • DecylTPP lowers mitochondrial ATP generation. • DecylTPP alters hydroethidine oxidation. [ABSTRACT FROM AUTHOR]

Homology modeling of gastric H,K-ATPase based on the E2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835839) revealed the presence of a single high-affinity binding site for K+ and an E2 form-specific salt bridge between Glu820 (M6) and Lys791 (M5). In the E820Q mutant this salt bridge is no longer possible, and the head group of Lys791, together with a water molecule, fills the position of the K+ ion and apparently mimics the K+-filled cation binding pocket. This gives an explanation for the K+-independent ATPase activity and dephosphorylation step of the E820Q mutant (Swarts, H. G. P., Hermsen, H. P. H., Koenderink, J. B., Schuurmans Stekhoven, F. M. A. H., and De Pont, J. J. H. H. M. (1998) EMBO J. 17, 3029-3035) and, indirectly, for its E1 preference. The model is strongly supported by a series of reported mutagenesis studies on charged and polar amino acid residues in the membrane domain. To further test this model, Lys791 was mutated alone and in combination with other crucial residues. In the K791A mutant, the K+ affinity was markedly reduced without altering the E2 preference of the enzyme. The K791A mutation prevented, in contrast to the K791R mutation, the spontaneous dephosphorylation of the E820Q mutant as well as its conformational equilibrium change toward E1. This indicates that the salt bridge is essential for high-affinity K+ binding and the E2 preference of H,K-ATPase. Moreover, its breakage (E820Q) can generate a K+-insensitive activity and an E1 preference. In addition, the study gives a molecular explanation for the electroneutrality of H,K-ATPases. [ABSTRACT FROM AUTHOR]

Enteroviruses modify several cellular functions to ensure efficient replication. However, some of these alterations can trigger a defensive apoptotic host-cell program. To prevent premature abortion of their productive cycle, enteroviruses have developed anti-apoptotic countermeasures. Here, we discuss recent evidence that the enterovirus 2B protein exerts an anti-apoptotic activity that is related to its ability to form pores in endoplasmic reticulum (ER) and Golgi membranes, thereby reducing their Ca2+ content and perturbing ER–mitochondrial Ca2+ signaling. [Copyright &y& Elsevier]

The coxsackievirus 2B protein is a small hydrophobic protein (99 amino acids) that increases host cell membrane permeability, possibly by forming homo-multimers that build membrane-integral pores. Previously, we defined the functional role of the two hydrophobic regions HR1 and HR2. Here, we investigated the importance of regions outside HR1 and HR2 for multimerization, increasing membrane permeability, subcellular localization, and virus replication through analysis of linker insertion and substitution mutants. From these studies, the following conclusions could be drawn. (i) The hydrophilic region (58RNHDD62) between HR1 and HR2 is critical for multimerization and increasing membrane permeability. Substitution analysis of Asn61and Asn62 demonstrated the preference for short polar side chains (Asp, Asn), residues that are often present in turns, over long polar side chains (Glu, Gln). This finding supports the idea that the hydrophilic region is involved in pore formation by facilitating a turn between HR1 and HR2 to reverse chain direction. (ii) Studies undertaken to define the downstream boundary of HR2 demonstrated that the aromatic residues Trp80 and Trp82, but not the positively charged residues Arg81, Lys84, and Lys86 are important for increasing membrane permeability. (iii) The N terminus is not required for multimerization but does contribute to the membrane-active character of 2B. (iv) The subcellular localization of 2B does not rely on regions outside HR1 and HR2 and does not require multimerization. (v) Virus replication requires both the membrane-active character and an additional function of 2B that is not connected to this activity. [ABSTRACT FROM AUTHOR]